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10.1245/ASO.2005.10.005
Annals of Surgical Oncology 12:733-742 (2005)
© 2005 Society of Surgical Oncology
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Original Article

Suppression of ß-Catenin by Antisense Oligomers Augments Tumor Response to Isolated Limb Perfusion in a Rodent Model of Adenomatous Polyposis Coli–Mutant Colon Cancer

Robert J. Canter, MD1, Susan B. Kesmodel, MD1, Daniel F. Heitjan, PhD2, Nirmal K. Veeramachaneni, MD3, Nahush A. Mokadam, MD3, Jeffrey A. Drebin, MD, PhD1 and Douglas L. Fraker, MD1

1 Department of Surgery, University of Pennsylvania School of Medicine, 3400 Spruce Street, 4 Silverstein, Philadelphia, Pennsylvania 19104
2 Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, 622 Blockley Hall, 423 Grandian Drive, Philadelphia, Pennsylvania 19104
3 Department of Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110

Correspondence: Address correspondence and reprint requests to: Douglas L. Fraker, MD; E-mail: frakerd{at}uphs.upenn.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Isolated hepatic perfusion has been used in patients with colorectal cancer (CRC) metastatic to the liver. We sought to determine whether perfusion with antisense oligodeoxynucleotides results in the downregulation of ß-catenin and whether this improves tumor response to isolated limb perfusion (ILP) in a heterotopic model of human CRC.

Methods: Adenomatous polyposis coli–mutant human CRC xenografts were implanted into athymic rats. Animals were randomized to the following groups: (1) no treatment, (2) control ILP, (3) melphalan ILP, (4) ILP with antisense specific for ß-catenin, (5) ILP with nonspecific antisense, and (6) melphalan plus ß-catenin–specific antisense ILP. Tumor response and Western blot analysis of protein expression were evaluated.

Results: The maximal decrease (mean ± SE) in tumor volume was 0% ± 10% for no treatment, 19% ± 14% for control ILP, 58% ± 3% for melphalan ILP, 58% ± 9% for ß-catenin–specific ILP, 13% ± 19% for nonspecific antisense ILP, and 73% ± 6% for melphalan plus ß-catenin–specific ILP (P < .05 for melphalan ILP, ß-catenin–specific ILP, and melphalan plus antisense ILP). Tumor regrowth was delayed for 6 days after control ILP, 24 days after melphalan ILP, 20 days after ß-catenin–specific ILP, 10 days after nonspecific antisense ILP, and 60 days after melphalan plus ß-catenin–specific ILP (P < .05 for melphalan plus ß-catenin–specific ILP compared with all others). Western blotting revealed prolonged suppression of ß-catenin expression after ß-catenin–specific ILP.

Conclusions: Short-term ß-catenin antisense treatment improves tumor response rates after ILP in a rodent model of human CRC.

Key Words: Isolated limb perfusion • ß-Catenin antisense • Adenomatous polyposis coli • Hepatic metastases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal cancer (CRC) metastatic to the liver is a significant clinical problem that affects approximately 50,000 patients per year. Without treatment, survival is typically measured in months, and liver failure contributes substantially to patient morbidity and mortality. Although surgical resection offers the only chance for cure, most patients have unresectable disease because of the number of lesions, their proximity to major vascular structures, advanced preexisting liver disease, or concomitant systemic metastases. Considerable attention has been directed toward improving treatment for patients with unresectable disease. Liver-directed therapies have shown promise for limiting the progression of hepatic disease and, in some cases, for extending survival.1–3

One technique of regional surgical therapy that has been applied to unresectable metastatic CRC is isolated hepatic perfusion (IHP). IHP is a technically challenging procedure that requires complete vascular control of the liver through cannulation of the hepatic artery and inferior vena cava with concurrent venovenous bypass. Vascular isolation of the liver allows the delivery and recirculation of high-dose chemotherapy to the tumor tissue with minimal systemic toxicity. Phase I and II clinical trials of IHP have been performed with melphalan and tumor necrosis factor as a corollary to studies of isolated limb perfusion (ILP) with this combination of agents for the treatment of advanced melanoma of the extremities.4–6 These studies have achieved responses rates of 75% to 85%, with occasional complete responses, even among heavily pretreated patients. However, median survival in these series remains in the range of 12 to 24 months. Thus, despite these overall favorable results, methods to optimize the response to IHP could benefit a substantial number of patients.

Mutations in the adenomatous polyposis coli (APC) gene have been shown to play a causal role in the development of both familial and sporadic cases of colorectal neoplasia, and approximately 80% of sporadic cases of CRC have been linked to APC mutations.7–9 Although the APC tumor-suppressor protein product has multiple cellular functions, including effects on cell-cycle regulation and apoptosis, the key oncogenic effect of mutated APC is widely believed to be the failure to degrade ß-catenin, with its resultant overexpression.7,10 This hypothesis is further supported by data showing that approximately 50% of human CRC tumors that have wild-type APC have gain-of-function ß-catenin mutations that lead to its overexpression.8,11 Therefore, selective downregulation of ß-catenin may be an effective therapeutic approach in the treatment of CRC.12,13

Antisense treatment with oligodeoxyribonucleotides (ODNs) complementary to a specific segment of messenger RNA has been used experimentally to inhibit the expression of specific gene products. Significant laboratory data have demonstrated antisense therapy to be effective in both tissue culture and preclinical studies.14 However, clinical translation in human trials has been hindered by rapid systemic degradation and elimination of the compounds, largely by renal excretion, with the subsequent need to treat patients with large doses of antisense agents for extended periods of time to achieve downregulation of the target protein.14 Because regional perfusion is predicated on vascular isolation of the target tissue to circulate high-dose chemotherapeutics, we hypothesized that antisense therapy would be particularly suited to perfusion techniques. Although IHP has not proven to be feasible in rodents, we and others have shown that ILP is and that results with conventional agents reproduce those seen clinically.15,16 Consequently, we sought to determine whether the short-term administration of antisense ODNs targeting ß-catenin could downregulate the ß-catenin protein in tumor xenografts and thereby improve tumor responses to ILP in a heterotopic model of APC-mutant human CRC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Line
The human APC-mutated colon adenocarcinoma cell line SW480 was obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in tissue culture with McCoy’s medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, penicillin G, streptomycin, amphotericin, and gentamicin.

Materials
Melphalan, obtained from GlaxoSmithKline (Research Triangle Park, NC), was initially dissolved in sterile diluent solution provided by the manufacturer and then diluted in ILP perfusate solution to a final concentration of 13 µg/mL rat limb volume. Matrigel basement membrane matrix was obtained from BD Biosciences (Palo Alto, CA) and stored in 1-mL aliquots for use in tumor inoculation.

Antisense Oligodeoxynucleotides
ß-Catenin–specific and –nonspecific phosphorothioate oligomers, 20 base pairs long, were obtained from Trilink Biotechnology (San Diego, CA). These sequences were chosen on the basis of extensive in vitro experiments performed by Roh et al.12 that demonstrated the selective and dose-dependent downregulation of ß-catenin expression at both the messenger RNA and protein level with the ß-catenin–specific sequence, without any corresponding effect from the nonspecific oligomer. In addition, no other complementary Watson-Crick base pairing was observed between these oligomers and any other gene product according to the published sequence of the human genome. Sequences of the oligomers were as follows: specific, TAAGA GCTTAACCACAACTG; nonspecific, TGAGAG CCTAACTACAATTA. Lyophilized oligomers received from the manufacturer were reconstituted in filter-sterilized distilled water to a concentration of 1 mg/mL and stored as individual aliquots at –40°C.

Animals and Tumor Cell Implantation
Female homozygous nude rats, aged 5 to 7 weeks, were obtained from the National Cancer Institute (Frederick, MD) and housed under standard light and accommodation conditions. To optimize tumor cell implantation, animals were subjected to 5 Gy of total body irradiation by using a cesium-137 irradiator (Atomic Energy of Canada Ltd., Ontario, Canada) 1 to 2 days before tumor inoculation. For tumor inoculation, animals were anesthetized with a combination of intraperitoneal (IP) ketamine 62.5 mg/kg body weight (Abbott Laboratories, Chicago, IL) and IP xylazine 5 mg/kg body weight (Webster Veterinary Supply, Sterling, MA).

SW480 tumor cells were harvested from 80% confluent cell culture conditions, counted, and resuspended in medium at a concentration of 100 x 106/ mL. The cells were diluted 1/1 in ice-cold Matrigel to a concentration of 50 x 106/mL. A total of 5 x 106 cells in 100-µL aliquots were then injected subcutaneously into the lateral aspect of the distal hind limbs of the animals. This regimen reliably produced 12- to 15-mm elliptical subcutaneous tumors approximately 4 weeks after inoculation. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

ILP Technique and Measurement of Tumor Volume
When tumors reached 12 to 15 mm in diameter, three to five animals per group were randomized to the following treatments: (1) no treatment, (2) control ILP with hetastarch solution, (3) melphalan ILP, (4) antisense ILP with a 20–base pair phosphorothioate oligomer specific for ß-catenin at a concentration of 5 mg/kg total body weight, (5) antisense ILP with a nonspecific oligomer, and (6) melphalan plus ß-catenin–specific antisense ILP.

ILP was performed as described previously.15,17 In brief, animals were anesthetized with IP ketamine (125 mg/kg) and xylazine (10 mg/kg), and the right groin was cleansed with povidone-iodine solution. A 3-cm incision was made, and the femoral artery and vein were exposed. A tourniquet was then loosely placed inferior to the inguinal ligament, and 80 U of heparin (Abbott Laboratories, Chicago, IL) was injected IP. With use of an operating microscope, 6-0 silk ties were placed to obtain proximal and distal control of the vessels. Perfusate consisting of 6 mL of 6% hetastarch in .9% sodium chloride (Abbott Laboratories) plus 80 U of heparin, with or without melphalan, was then added to a glass oxygenating reservoir (Radnoti Glass Technology Incorporated, Monrovia, CA), and the system was primed. Veterinary silastic tubing was used to cannulate the vessels (arterial, .30-mm inner diameter and .64-mm outer diameter; venous, .64-mm inner diameter and 1.19-mm outer diameter; Konigsberg Instruments, Pasadena, CA), and after cannulation, the tourniquet was tightened. Venous drainage flowed by gravity into the reservoir, and arterial inflow was infused by a rotary pump (Masterflex 7524-00; Bar-nant Corporation, Barrington, IL) at 1.5 mL/min.

Normothermic ILP was then performed for 10 minutes, followed by a 2-minute washout perfusion with hetastarch solution. The pump was then stopped, the tourniquet was loosened, and the femoral cannulas were removed. The vessels were then ligated with 6-0 silk ties, the incision was sutured with 4-0 Vicryl (Ethicon Incorporated, Somerville, NJ), and the animals were allowed to recover from anesthesia.

Response to treatment was observed for 60 days unless large tumors (i.e., diameter >30 mm) were present that necessitated animal death in accordance with animal care protocols. Tumors were measured with calipers every 3 days, and tumor volume was calculated by using a formula (length x width2 x .52) that approximates the volume of an elliptical solid. The volume of full-thickness eschar or ulceration of tumors was similarly measured and subtracted from the overall volume to estimate viable tumor volume. The percentage change in tumor volume was determined by normalizing to the starting volume at the time of ILP.

Protein Extraction and Western Blot Analysis
At selected time points after ILP, animals were anesthetized with a combination of IP ketamine 62.5 mg/kg body weight (Abbott Laboratories) and IP xylazine 5 mg/kg body weight (Webster Veterinary Supply). Tumors were then completely excised, washed in ice-cold phosphate-buffered saline, and homogenized in an ice-cold lysis buffer containing 20 mM of HEPES (pH 7.2), 50 mM of NaCl, 1 mM of EDTA, 1 mM of EGTA, 1 mM of phenylmethylsulfonyl fluoride, 2 µg/mL of aprotinin, 2 µg/mL of leupeptin, and 2 µg/mL of pepstatin. Insoluble tissue was removed by centrifugation at 2000 x g for 10 minutes. The soluble fraction was then incubated on ice for 15 minutes with 1% Triton X-100. After repeat centrifugation at 30,000 x g for 30 minutes, individual protein aliquots were stored at –80°C for subsequent gel electrophoresis. Quantitation of protein was performed with a BCA Protein Assay kit (Pierce, Rockford, IL).

For Western blotting, 30 µg of protein from each time point was electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide electrophoresis gel and transferred to polyvinylidene difluoride membranes. Membranes were then blocked for 1 hour at room temperature with 5% nonfat milk in Tris-buffered saline (TBS). Polyclonal rabbit antibodies specific for ß- and {gamma}-catenin, obtained from Santa Cruz Biotechnology (Santa Cruz, CA), were added for 1 hour at room temperature at dilutions of 1/4000 and 1/1000 for ß- and {gamma}-catenin, respectively. Blots were then washed with TBS containing .1% Tween 20 and then incubated for 1 hour at room temperature with a polyclonal horseradish peroxidase–conjugated goat anti-rabbit antibody at 1/4000 dilution in 5% nonfat milk in TBS. An enhanced chemiluminescence kit (Amersham, Piscataway, NJ) was used to detect immunoreactive bands. After detection of ß- and {gamma}-catenin protein bands, blots were washed in TBS containing .1% Tween 20, reblocked with 5% nonfat milk in TBS, and reprobed at 1/1000 dilution with a polyclonal rabbit goat anti-actin antibody (Santa Cruz Biotechnology) to confirm equal loading of protein. All Western blots were repeated 3 times, and representative blots are shown. Animals used for Western blot analysis of protein expression were sacrificed after collection of tumor tissue and therefore constituted a separate experimental group.

Statistical Analysis
Absolute tumor volume measurements were transformed to a logarithmic scale to improve the normality and homogeneity of the error variances between measurements.18 Logarithmic transformation also enabled the slopes of the data sets to be interpreted as growth rates because they achieved an approximately linear pattern. Tumor growth was then modeled with mixed linear models that assumed that the animals were independent but that serial tumor volumes for each individual rodent were correlated. Analysis of the data in this way suggested that in each treatment group, there was an initial decline in tumor volume that lasted approximately 10 days, followed by a relatively static period of growth between 11 and 25 days and then a more exponential pattern of growth after 25 days. Therefore, we modeled the tumor growth curves on the basis of a linear spline with transition points at days 10 and 25.19 This assumed that growth progressed with one slope until day 10, at which point the slope changed to another value that remained constant for the next observation period, which then changed to a different constant value for the final period. Differences in tumor volume were thus analyzed by comparing declining slopes in the early period, before day 10, and increasing slopes in the later periods, between days 11 and 25, and after day 25. Analyses were performed by using SAS MIXED (SAS Institute, Cary, NC), and a P value ≤.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor Response After ILP With Melphalan
We have previously shown that melanoma xeno-grafts in the nude rat respond to experimental ILP in a manner that reproduces the results seen clinically.15 In particular, ILP with melphalan leads to an approximately 75% reduction in the volume of established tumors and delayed regrowth relative to control perfusions, analogous to a partial response. Because IHP is too technically complex to perform in rodents, we chose to test our melphalan ILP model against heterotopic APC-mutant human CRC xenografts grown in the hind limbs of nude rats. Figure 1Go depicts the relative change in tumor volume after ILP over 60 days. The maximal decrease (mean ± SE) in viable tumor volume relative to tumor starting volume was 0% ± 10% for animals that underwent no treatment, 19% ± 14% for animals that underwent control ILP, and 58% ± 3% for animals that underwent melphalan ILP (P < .05 for melphalan ILP compared with no treatment and control ILP). In addition, tumor re-growth was delayed 0 days for animals that received no treatment versus 6 days after control ILP and 24 days after melphalan ILP (Fig. 1Go).


Figure 1
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  		FIG. 1. Percentage change in relative tumor volume over time comparing tumor response after no treatment, control isolated limb perfusion (ILP), and melphalan ILP. Data are expressed as viable tumor volume relative to starting tumor volume (mean ± SEM) for 3 to 5 animals per group.

 
This 60% reduction in tumor size on average after melphalan ILP relative to no treatment, in conjunction with a 24-day delay in tumor regrowth, is also consistent with a partial response to treatment. This finding is similar to those in most patients who undergo hepatic perfusion for metastatic CRC.4–6 Thus, although we used a heterotopic model of regional perfusion for metastatic CRC, our results with melphalan alone suggest that it is a valid model for studying the response of CRC xenografts to isolation perfusion.

Tumor Response After ILP With ß-Catenin – Specific and –Nonspecific Antisense Treatment
Figure 2Go shows the response rates to ILP with the addition to the perfusate of both specific and nonspecific antisense oligomers targeted to ß-catenin messenger RNA. Our sequences are based on the work of Roh et al.12 which demonstrated selective downregulation in vitro and in vivo of both ß-catenin messenger RNA and protein with the specific sequence, compared with no effect with the nonspecific sequence. In addition, no other interactions were apparent between either oligomer and any other gene sequence in the published human genome. For ILP, antisense oligomers were added to the perfusate at a dose of 5 mg/kg total body weight according to in vivo data from Roh et al. with similar doses, as well as preliminary dose-response studies from our own laboratory (data not shown) that showed effective downregulation of ß-catenin protein at a dose of 5 mg/kg.


Figure 2
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  		FIG. 2. Percentage change in relative tumor volume over time comparing tumor response after no treatment, control isolated limb perfusion (ILP), melphalan ILP, and ILP with ß-catenin–specific and –nonspecific anti-sense oligomers. Data are expressed as viable tumor volume relative to starting tumor volume (mean ± SEM) for 3 to 5 animals per group.

 
The maximal decrease (mean ± SE) in viable tumor volume relative to tumor starting volume was 58% ± 9% for ß-catenin–specific ILP and 13% ± 19% for nonspecific oligomer ILP (P < .05). Tumor regrowth was delayed for 20 days after ß-catenin–specific ILP, compared with 10 days after nonspecific oligomer ILP. Although there was a minimal tumor response and subsequent delay in tumor regrowth after ILP with nonspecific antisense, this was not significantly different from that observed after control ILP. In contrast, the increased tumor regression and tumor growth delay after ß-catenin–specific antisense ILP supports the hypothesis that targeted downregulation of ß-catenin expression leads to an antitumor effect.

Additive Response After ILP With Melphalan and ß- Catenin – Specific Antisense Oligomers
With the combination of melphalan and ß-catenin–specific antisense ILP (Fig. 3Go), the maximal decrease (mean ± SE) in viable tumor volume relative to tumor starting volume was 73% ± 6% (P < .05 compared with control ILP and nonspecific antisense ILP, but P > .05 compared with melphalan alone and ß-catenin–specific antisense oligomer alone). Tumor regrowth with the combination of melphalan and ß-catenin–specific antisense was delayed 60 days—significantly longer than all the other treatment groups (P < .05 compared with all groups). These data suggest that the combination of melphalan ILP with ß-catenin–specific antisense has an additive effect against APC-mutant CRC xenografts, particularly with respect to delaying tumor regrowth. Figure 4Go shows pictures of representative tumors 7 days after treatment for each of the groups. The improved response from the addition of ß-catenin–specific antisense to standard melphalan ILP can be seen.


Figure 3
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  		FIG. 3. Percentage change in relative tumor volume over time comparing tumor response after no treatment, control isolated limb perfusion (ILP), melphalan ILP, ß-catenin–specific antisense ILP, and combined melphalan/ß-catenin–specific antisense ILP. Data are expressed as viable tumor volume relative to starting tumor volume (mean ± SEM) for 3 to 5 animals per group.

 

Figure 4
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  		FIG. 4. Pictures of representative tumors from each group 7 days after randomization and treatment. ILP, isolated limb perfusion.

 
Changes in Protein Expression After ILP
Western blot analysis revealed a significant, near-complete downregulation of both ß-catenin and {gamma}-catenin expression within 24 hours of control ILP (Fig. 5AGo). This was followed by a return to pretreatment levels of both proteins by post-ILP day 7. After ILP with nonspecific antisense oligomer (Fig. 5BGo), ß- and {gamma}-catenin expression display a similar pattern of downregulation consistent with an effect from the ILP procedure itself rather than a nonspecific effect from random antisense effects on the tumor.


Figure 5
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  		FIG. 5. Representative Western blot analyses (from 3 independent replicates) demonstrating ß-catenin, {gamma}-catenin, and actin protein expression at selected time points after control isolated limb perfusion (ILP) (A), nonspecific antisense ILP (B), and ß-catenin–specific antisense ILP (C). Animals used for Western blot analysis were sacrificed after collection of tumor tissue and, therefore, constituted a separate experimental group. POD, postoperative day.

 
In contrast, Fig. 5CGo demonstrates that ILP with ß-catenin–specific oligomers leads to a more prolonged suppression of ß-catenin expression in the tumors, with protein expression only starting to return 14 days after ILP. Conversely, the pattern of {gamma}-catenin expression after ß-catenin–specific ILP resembles that after control and nonspecific antisense ILP (i.e., a near-complete loss of {gamma}-catenin expression within 24 hours but a return to pretreatment levels by post-ILP day 7). These data demonstrate that treatment with the ß-catenin–specific antisense oligomer leads to a minimum of seven additional days of selective downregulation of ß-catenin protein expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CRC metastatic to the liver remains a significant clinical problem with considerable morbidity and mortality. Although surgical resection remains the mainstay of therapy, a significant number of patients have unresectable disease. For these patients, treatment options are limited. Regional liver-directed therapies have received attention in an attempt to improve patient outcomes. Although results with these modalities have been encouraging, strategies to optimize these techniques could benefit a substantial number of patients.

IHP is a highly specialized surgical technique that requires complete vascular isolation of the liver during which the treatment agents are recirculated through an extracorporeal pump oxygenator bypass circuit. This enables the dose of therapeutic agents to be markedly escalated becauseexposure to the organs of metabolism and toxicity is limited. IHP is an ideal technique for the application of antisense technology because previous clinical trials with systemic antisense agents have been hindered by rapid clearance and poor intracellular delivery of the drugs. These pharmacokinetic limitations have led some investigators in clinical trials to use extremely high doses of parenterally delivered antisense oligomers to achieve target gene suppression.14 However, this strategy has been complicated by increased toxicity and cost. IHP can circumvent the problems of rapid clearance observed with systemic administration of antisense oligomers and can allow delivery of reduced doses of antisense agents directly to the target tissue. In addition, the delivery of antisense compounds by regional perfusion may increase the efficacy of the agents because extracorporeal perfusion (e.g., cardio-pulmonary bypass) has demonstrated promise for increasing the uptake of virally mediated transfection vectors in preclinical models of gene therapy.20

Our data demonstrate that ILP in athymic rats is a valid laboratory model for evaluating tumor response rates after regional perfusion of human CRC xenografts. Although the tumors are implanted in a heterotopic location and the limb is perfused, rather than the liver, established tumors grown in rodent hind limbs regress and demonstrate delayed regrowth after melphalan ILP in a pattern analogous to the partial responses seen in clinical trials of IHP. Because this model does not involve perfusion of the liver, we cannot evaluate potential hepatic toxicity, which is an important concern when genetic material is used. Nevertheless, it seems worthwhile to have a relevant preclinical model of metastatic CRC that permits testing and optimization of tumor response rates after regional perfusion.

We have also shown that ILP with antisense oligomers specifically targeted to downregulate ß-catenin expression causes partial tumor regression (58% by volume) and delays tumor regrowth (20 days) of CRC xenografts that have APC mutations and therefore overexpress ß-catenin. Conversely, when ILP with a nonspecific antisense oligomer is performed, tumor response is similar to that after control ILP. These results reinforce the principle that suppression of ß-catenin can have significant anti-neoplastic activity in tumors that are APC mutant, and they demonstrate that short-term exposure to high-concentration antisense ODNs can result in substantial suppression of the antisense target. It remains to be determined whether suppression of ß-catenin in the absence of APC mutations, i.e., when either gain-of-function ß-catenin mutations or wild-type ß-catenin is present, will also have antitumor effects.

Although Western blot analysis demonstrated downregulation of both ß- and {gamma}-catenin for 7 days after both control and nonspecific antisense ILP, the ß-catenin–specific antisense oligomer selectively suppressed ß-catenin expression for a significantly longer duration; only a weak signal was present 14 days after ILP. This result supports a specific anti–ß-catenin effect from the ß-catenin antisense oligomer. Although the changes in ß- and {gamma}-catenin protein expression observed in the first 7 days after control and nonspecific antisense ILP remain unexplained, it is important to note that the suppression of these proteins occurs with control ILP and therefore does not reflect a nonspecific or nonselective effect of antisense treatment against ß-catenin.

In our limb perfusion model, unlike other groups performing rodent ILP,16,21 we have been unable to successfully reconstruct the femoral vessels with any significant patency after cannulation and perfusion. This is likely because the animals in our system are, on average, 20% to 25% smaller than those of the other investigators. Therefore, all animals in our experiments that undergo any form of ILP are subjected to ligation of the femoral vessels. Consequently, we have previously hypothesized that the minimal tumor response seen after control ILP is secondary to limb ischemia. Although this introduces a potentially confounding factor regarding the effect of ischemia on tumor response after control ILP, this minimal response remains statistically significantly less than the response observed after melphalan, ß-catenin–specific, or combined melphalan and ß-catenin–specific ILP.

It is also possible that the early suppression of ß- and {gamma}-catenin expression after control and nonspecific antisense ILP reflects the effects of ischemia. Further studies should help to clarify whether there is a generalized reduction in protein expression in the tumor tissue secondary to the ILP procedure itself or whether ß- and {gamma}-catenin are selectively affected during this early period after perfusion. Nevertheless, the specific suppression of ß-catenin for an additional period of days after treatment with the ß-catenin–specific oligomer in combination with the augmented tumor response rates with this therapy suggest that the mechanism for the increased response is through the specific suppression of ß-catenin. Although the implications of the early downregulation of {gamma}-catenin after control ILP remain unexplained, it is important to note that there is a paucity of evidence linking {gamma}-catenin to pro-oncogenic activity and that no {gamma}-catenin mutations have been reported in human cancers.22 This is in contrast to the extensive literature implicating ß-catenin as a proto-oncogene.23

When ß-catenin–specific antisense oligomers were combined with melphalan ILP, there was an additive antitumor effect with a significantly greater reduction in tumor volume (73%) than the other treatment groups and a significantly longer delay in tumor re-growth (60 days). The mechanism for this augmented effect remains undetermined, particularly because ß-catenin expression returns to normal levels between 14 and 30 days after ILP. Melphalan is an alkylating agent that is known to interfere with DNA synthesis by cross-linking DNA strands. ß-Catenin has multiple cellular functions, including regulation of the T-cell factor transcription factor/Myc oncogene pathway and the cadherin/cell adhesion complex.24–28 Recent evidence also suggests that ß-catenin overexpression is proangiogenic.29 Although we have not yet determined the mechanism for the effects described here, our data nonetheless demonstrate that antisense ODN treatment is effective in a regional perfusion model and that the combination of ß-catenin suppression and melphalan chemotherapy leads to a significant and prolonged tumor growth delay.

In summary, specific suppression of ß-catenin by antisense oligomers augments tumor response rates to ILP in a rodent model of APC-mutant colon cancer. Selective downregulation of ß-catenin with antisense oligomers may therefore hold promise as an additional therapeutic modality in the regional treatment of patients with CRC metastatic to the liver.


   ACKNOWLEDGMENTS
 
Supported by the Georgene S. Harmelin Endowment Fund (D.L.F.) and by grants from the National Institutes of Health and the Burroughs Wellcome Fund (J.A.D.).


   FOOTNOTES
 
Presented in part at the Surgical Forum of the American College of Surgeons, Chicago, Illinois, October 19–23, 2003.

Received for publication October 13, 2004. Accepted for publication May 8, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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